This invention relates to nucleic acid molecules with catalytic activity and derivatives thereof.
The following is a brief description of enzymatic nucleic acid molecules. This summary is not meant to be complete but is provided only for understanding of the invention that follows. This summary is not an admission that all of the work described below is prior art to the claimed invention.
Enzymatic nucleic acid molecules (ribozymes) are nucleic acid molecules capable of catalyzing one or more of a variety of reactions, including the ability to repeatedly cleave other separate nucleic acid molecules in a nucleotide base sequence-specific manner. Such enzymatic nucleic acid molecules can be used, for example, to target virtually any RNA transcript (Zaug et al., 324, Nature 429 1986; Cech, 260 JAMA 3030, 1988; and Jefferies et al., 17 Nucleic Acids Research 1371, 1989).
Because of their sequence-specificity, trans-cleaving enzymatic nucleic acid molecules show promise as therapeutic agents for human disease (Usman and McSwiggen, 1995 Ann. Rep. Med. Chem. 30, 285-294; Christoffersen and Marr, 1995 J. Med. Chem. 38, 2023-2037). Enzymatic nucleic acid molecules can be designed to cleave specific RNA targets within the background of cellular RNA. Such a cleavage event renders the mRNA non-functional and abrogates protein expression from that RNA. In this manner, synthesis of a protein associated with a disease state can be selectively inhibited.
There are seven basic varieties of naturally occurring enzymatic RNAs. Each can catalyze the hydrolysis of RNA phosphodiester bonds in trans (and thus can cleave other RNA molecules) under physiological conditions. In general, enzymatic nucleic acids act by first binding to a target RNA. Such binding occurs through the target binding portion of a enzymatic nucleic acid which is held in close proximity to an enzymatic portion of the molecule that acts to cleave the target RNA. Thus, the enzymatic nucleic acid first recognizes and then binds a target RNA through complementary base pairing, and once bound to the correct site, acts enzymatically to cut the target RNA. Strategic cleavage of such a target RNA will destroy its ability to direct synthesis of an encoded protein. After an enzymatic nucleic acid has bound and cleaved its RNA target, it is released from that RNA to search for another target and can repeatedly bind and cleave new targets.
In addition, several in vitro selection (evolution) strategies (Orgel, 1979, Proc. R. Soc. London, B 205, 435) have been used to evolve new nucleic acid catalysts capable of catalyzing a variety of reactions, such as cleavage and ligation of phosphodiester linkages and amide linkages, (Joyce, 1989, Gene, 82, 83-87; Beaudry et al., 1992, Science 257, 635-641; Joyce, 1992, Scientific American 267, 90-97; Breaker et al., 1994, TIBTECH 12, 268; Bartel et al., 1993, Science 261:1411-1418; Szostak, 1993, TIBS 17, 89-93; Kumar et al, 1995, FASEB J., 9, 1183; Breaker, 1996, Curr. Op. Biotech., 7, 442).
The enzymatic nature of a ribozyme is advantageous over other technologies, since the effective concentration of ribozyme necessary to effect a therapeutic treatment is generally lower than that of an antisense oligonucleotide. This advantage reflects the ability of the ribozyme to act enzymatically, Thus, a single ribozyme (enzymatic nucleic acid) molecule is able to cleave many molecules of target RNA. In addition, the ribozyme is a highly specific inhibitor, with the specificity of inhibition depending not only on the base-pairing mechanism of binding, but also on the mechanism by which the molecule inhibits the expression of the RNA to which it binds. That is, the inhibition is caused by cleavage of the RNA target and so specificity is defined as the ratio of the rate of cleavage of the targeted RNA over the rate of cleavage of non-targeted RNA. This cleavage mechanism is dependent upon factors additional to those involved in base pairing. Thus, it is thought that the specificity of action of a ribozyme is greater than that of antisense oligonucleotide binding the same RNA site.
The development of ribozymes that are optimal for catalytic activity would contribute significantly to any strategy that employs RNA-cleaving ribozymes for the purpose of regulating gene expression. The hammerhead ribozyme functions with a catalytic rate (kcat) of xcx9c1 minxe2x88x921 in the presence of saturating (10 mM) concentrations of Mg2+ cofactor. However, the rate for this ribozyme in Mg2+ concentrations that are closer to those found inside cells (0.5-2 mM) can be 10- to 100-fold slower. In contrast, the RNase P holoenzyme can catalyze pre-tRNA cleavage with a kcat of xcx9c30 minxe2x88x921 under optimal assay conditions. An artificial xe2x80x98RNA ligasexe2x80x99 ribozyme has been shown to catalyze the corresponding self-modification reaction with a rate of xcx9c100 minxe2x88x921. In addition, it is known that certain modified hammerhead ribozymes that have substrate binding arms made of DNA catalyze RNA cleavage with multiple turn-over rates that approach 100 minxe2x88x921. Finally, replacement of a specific residue within the catalytic core of the hammerhead with certain nucleotide analogues gives modified ribozymes that show as much as a 10-fold improvement in catalytic rate. These findings demonstrate that ribozymes can promote chemical transformations with catalytic rates that are significantly greater than those displayed in vitro by most natural self-cleaving ribozymes. It is then possible that the structures of certain self-cleaving ribozymes may be optimized to give maximal catalytic activity, or that entirely new RNA motifs can be made that display significantly faster rates for RNA phosphodiester cleavage.
An extensive array of site-directed mutagenesis studies have been conducted with the hammerhead ribozyme to probe relationships between nucleotide sequence and catalytic activity. These systematic studies have made clear that most nucleotides in the conserved core of the hammerhead ribozyme cannot be mutated without significant loss of catalytic activity. In contrast, a combinatorial strategy that simultaneously screens a large pool of mutagenized ribozymes for RNAs that retain catalytic activity could be used more efficiently to define immutable sequences and to identify new ribozyme variants. Although similar in vitro selection experiments have been conducted with the hammerhead ribozyme (Nakamaye and Eckstein, 1994, Biochemistry 33, 1271; Long and Uhlenbeck, 1994, Proc. Natl. Acad. Sci., 91, 6977; Ishizaka et al., 1995, BBRC 214, 403; Vaish et al., 1997, Biochemistry 36, 6495), none of these efforts have successfully screened full-sized hammerhead ribozymes for all possible combinations of sequence variants that encompass the entire catalytic core.
The hammerhead ribozyme is one of the smallest ribozymes known and has thus attracted much attention for the study of structure-function relationships in catalytic. RNAs as well as for its potential for the sequence-specific inhibition of gene expression (Usman et al., supra). The hammerhead cleaves RNA sequence-specifically adjacent to the general triplet sequence NUX, where N is any nucleotide and X can be A, U or C. Cleavage behind a guanosine such as in GUG is very slow (4.3xc3x9710xe2x88x925 minxe2x88x921) compared to the triplet substrate GUC (1 minxe2x88x921) (Baidya et al., 1997, Biochemistry 36, 1108). Although the X-ray structure of this ribozyme has been solved and a mechanism proposed (Pley et al., 1994 Nature, 372, 68; Scott et al., 1996, Science 274, 2065), the question of what determines its specificity for the NUX sequence is still largely unresolved. One way of obtaining an insight into this problem might be to compare sequences of hammerhead ribozymes with different triplet cleaving specificities. In previous publications, it was demonstrated, using in vitro selection, that the native hammerhead sequence necessary to cleave a typical cleavage triplet, NUX, can not be altered (Nakamaye and Eckstein, 1994, Biochemistry 33, 1271; Long and Uhlenbeck, 1994, Proc. Natl. Acad. Sci., 91, 6977; Ishizaka et al., 1995, BBRC 214, 403; Vaish et al., 1997, Biochemistry 36, 6495), indicating that nature has evolved a close to perfect GUC cleaver. It was of interest to see what changes have to be imposed on the native hammerhead sequence for it to cleave after AUG, which usually resists cleavage, and thus arrive at a ribozyme with a new specificity. To achieve this, an in vitro selection was undertaken, where the number of randomized positions in the starting pool corresponded to the number of nucleotides typically found in the core and stem-loop II region of a hammerhead ribozyme. This would allow all possible sequence permutations to be explored in the search for a hammerhead which is able to cleave behind AUG (Robertson and Joyce, 1990, Nature 344:6265 467-8; Ellington and Szostak, 1990; Tuerk and Gold, 1990; Wright and Joyce, 1997; Carmi et al., 1997; for reviews see Breaker, 1997; Abelson, 1996; Ellington, 1997; Chapman and Szostak, 1994). Tang et al., 1997, RNA 3, 914, reported novel ribozyme sequences with endonuclease activity, where the authors used an in vitro selection approach to isolate these ribozymes.
The platelet derived growth factor (PDGF) system has served as a prototype for the identification of substrates of receptor tyrosine kinases. Certain enzymes become activated by the PDGF receptor kinase, including phospholipase C and phosphatidylinositol 3xe2x80x2 kinase, Ras guanosine triphosphate (GTPase) activating protein (GAP) and src-like tyrosine kinases. GAP regulates the function of the Ras protein. It stimulates the GTPase activity of the 21 kD Ras protein (Barbacid. 56 Ann. Rev. Biochem. 779, 1987). Microinjection of oncogenically activated Ras into NIH 3T3 cells induces DNA synthesis. Mutations that cause oncogenic activation of ras lead to accumulation of Ras bound to GTP, the active form of the molecule. These mutations block the ability of GAP to convert Ras to the inactive form. Mutations that impair the interactions of Ras with GAP also block the biological function of Ras.
While a number of ras alleles exist (N-ras, K-ras, H-ras) which have been implicated in carcinogenesis, the type most often associated with colon and pancreatic carcinomas is the K-ras. Ribozymes which are targeted to certain regions of the K-ras allelic mRNAs may also prove inhibitory to the function of the other allelic mRNAs of the N-ras and H-ras genes.
Transformation is a cumulative process whereby normal control of cell growth and differentiation is interrupted, usually through the accumulation of mutations affecting the expression of genes that regulate cell growth and differentiation.
Scanlon WO91/18625, WO91/18624, and WO91/18913 describe a specific hammerhead ribozyme effective to cleave RNA from the H-ras gene at codon 12 mutation. This ribozyme is said to inhibit H-ras expression in response to exogenous stimuli.
Thompson et al., U.S. Pat. Nos. 5,610,052 and 5,801,158, describes enzymatic RNA molecules against Ras.
Vaish et al., 1998 PNAS 95, 2158-2162, describes the in vitro selection of a hammerhead-like ribozyme with an extended range of cleavable triplets.
The references cited above are distinct from the presently claimed invention since they do not disclose and/or contemplate the catalytic nucleic acid molecules of the instant invention.
This invention relates to novel nucleic acid molecules with catalytic activity, which are particularly useful for cleavage of RNA or DNA. The nucleic acid catalysts of the instant invention are distinct from other nucleic acid catalysts known in the art. The nucleic acid catalysts of the instant invention do not share sequence homology with other known ribozymes. Specifically, nucleic acid catalysts of the instant invention are capable of catalyzing an intermolecular or intramolecular endonuclease reaction.
In a preferred embodiment, the invention features a nucleic acid molecule with catalytic activity having one of the formulae I-V: 
In each of the above formulae, N represents independently a nucleotide or a non-nucleotide linker, which may be the same or different; X and Y are independently oligonucleotides of length sufficient to stably interact (e.g., by forming hydrogen bonds with complementary nucleotides in the target) with a target nucleic acid molecule (the target can be an RNA, DNA or RNA/DNA mixed polymers); m and n are integers independently greater than or equal to 1 and preferably less than about 100, wherein if (N)m and (N)n are nucleotides, (N)m and (N)n are optionally able to interact by hydrogen bond interaction; J is an oligonucleotide of length 6 or 7 nucleotides; K is an oligonucleotide of length 5 nucleotides; L is a linker which may be present or absent (i.e., the molecule is assembled from two separate molecules), but when present, is a nucleotide and/or a non-nucleotide linker, which may be a single-stranded and/or double-stranded region; and xe2x80x94xe2x80x94xe2x80x94 represents a chemical linkage (e.g. a phosphate ester linkage, amide linkage or others known in the art). Z is independently a nucleotide numbered as 13.1 (FIG. 8) which can stably interact with a nucleotide at position 14.1 (FIG. 8) in the target nucleic acid molecule, preferably, the N13.1-N14.1 interaction is inosine13.1-cytidine14.1; adenosine13.1-uridine14.1; guanosine13.1-cytidine14.1; uridine13.1-adenosine14.1; or cytidine13.1-guanosine14.1; and C, G, A, and U represent cytidine, guanosine, adenosine and uridine nucleotides, respectively. The nucleotides in the each of the formulae I-V are unmodified or modified at the sugar, base, and/or phosphate as known in the art.
In a preferred embodiment, the invention features nucleic acid molecules of Formula I, where the sequence of oligonucleotide J is selected from the group comprising 5xe2x80x2-GGCAUCC-3xe2x80x2, 5xe2x80x2-AGCAUU-3xe2x80x2, 5xe2x80x2-GCAUCA-3xe2x80x2, 5xe2x80x2-AGCAUC-3xe2x80x2, and 5xe2x80x2-AGCGUC-3xe2x80x2.
In another preferred embodiment, the invention features nucleic acid molecules of Formula II, where the sequence of oligonucleotide J is 5xe2x80x2-GGAAUC-3xe2x80x2.
In a further preferred embodiment, the invention features nucleic acid molecules of Formula III, where the sequence of oligonucleotide K is selected from the group comprising 5xe2x80x2-UGAUG-3xe2x80x2, 5xe2x80x2-UGAUC-3xe2x80x2, and 5xe2x80x2-UGGGC-3xe2x80x2.
In another embodiment, the nucleotide linker (L) is a nucleic acid sequence selected from the group consisting of 5xe2x80x2-GCACU-3xe2x80x2, 5xe2x80x2-GAACU-3xe2x80x2, 5xe2x80x2-GCACC-3xe2x80x2, 5xe2x80x2-GNACU-3xe2x80x2, 5xe2x80x2-GNGCU-3xe2x80x2, 5xe2x80x2-GNCCU-3xe2x80x2, 5xe2x80x2-GNUCU-3xe2x80x2, 5xe2x80x2-GNGUU-3xe2x80x2, 5xe2x80x2-GNCUU-3xe2x80x2, 5xe2x80x2-GNUUU-3xe2x80x2, 5xe2x80x2-GNAUU-3xe2x80x2, 5xe2x80x2-GNACA-3xe2x80x2, 5xe2x80x2-GNGCA-3xe2x80x2, 5xe2x80x2-GNCCA-3xe2x80x2, and 5xe2x80x2-GNUCA-3xe2x80x2, whereby N can be selected from the group consisting of A, G, C, or U.
In yet another embodiment, the nucleotide linker (L) is a nucleic acid aptamer, such as an ATP aptamer, HIV Rev aptamer (RRE), HIV Tat aptamer (TAR) and others (for a review see Gold et al., 1995, Annu. Rev. Biochem., 64, 763; and Szostak and Ellington, 1993, in The RNA World, ed. Gesteland and Atkins, pp. 511, CSH Laboratory Press). A xe2x80x9cnucleic acid aptamerxe2x80x9d as used herein is meant to indicate a nucleic acid sequence capable of interacting with a ligand. The ligand can be any natural or a synthetic molecule, including but not limited to a resin, metabolites, nucleosides, nucleotides, drugs, toxins, transition state analogs, peptides, lipids, proteins, amino acids, nucleic acid molecules, hormones, carbohydrates, receptors, cells, viruses, bacteria and others.
In yet another embodiment, the non-nucleotide linker (L) is as defined herein. The term xe2x80x9cnon-nucleotidexe2x80x9d as used herein include either abasic nucleotide, polyether, polyamine, polyamide, peptide, carbohydrate, lipid, or polyhydrocarbon compounds. Specific examples include those described by Seela and Kaiser, Nucleic Acids Res. 1990, 18:6353 and Nucleic Acids Res. 1987, 15:3113; Cload and Schepartz, J. Am. Chem. Soc. 1991, 113:6324; Richardson and Schepartz, J. Am. Chem. Soc. 1991, 113:5109; Ma et al., Nucleic Acids Res. 1993, 21:2585 and Biochemistry 1993, 32:1751; Durand et al., Nucleic Acids Res. 1990, 18:6353; McCurdy et al., Nucleosides and Nucleotides 1991, 10:287; Jschke et al., Tetrahedron Lett. 1993, 34:301; Ono et al.; Biochemistry 1991, 30:9914; Arnold et al., International Publication No. WO 89/02439; Usman et al., International Publication No. WO 95/06731; Dudycz et al., International Publication No. WO 95/11910 and Ferentz and Verdine, J. Am. Chem. Soc. 1991, 113:4000, all hereby incorporated by reference herein. Thus, in a preferred embodiment, the invention features an enzymatic nucleic acid molecule having one or more non-nucleotide moieties, and having enzymatic activity to cleave an RNA or DNA molecule. By the term xe2x80x9cnon-nucleotidexe2x80x9d is meant any group or compound which can be incorporated into a nucleic acid chain in the place of one or more nucleotide units, including either sugar and/or phosphate substitutions, and allows the remaining bases to exhibit their enzymatic activity. The group or compound is abasic in that it does not contain a commonly recognized nucleotide base, such as adenine, guanine, cytosine, uracil or thymine. The terms xe2x80x9cabasicxe2x80x9d or xe2x80x9cabasic nucleotidexe2x80x9d as used herein encompass sugar moieties lacking a base or having other chemical groups in place of a base at the 1xe2x80x2 position.
In preferred embodiments, the enzymatic nucleic acid includes one or more stretches of RNA, which provide the enzymatic activity of the molecule, linked to the non-nucleotide moiety. The necessary RNA components are known in the art (see for e.g., Usman et al., supra). By RNA is meant a molecule comprising at least one ribonucleotide residue. By xe2x80x9cribonucleotidexe2x80x9d is meant a nucleotide with a hydroxyl group at the 2xe2x80x2 position of a xcex2-D-ribo-furanose moiety.
Thus, in one preferred embodiment, the invention features ribozymes that inhibit gene expression and/or cell proliferation. These chemically or enzymatically synthesized nucleic acid molecules contain substrate binding domains that bind to accessible regions of specific target nucleic acid molecules. The nucleic acid molecules also contain domains that catalyze the cleavage of target. Upon binding, the enzymatic nucleic acid molecules cleave the target molecules, preventing, for example, translation and protein accumulation. In the absence of the expression of the target gene, cell proliferation, for example, is inhibited. In another aspect of the invention, enzymatic nucleic acid molecules that cleave target molecules are expressed from transcription units inserted into DNA or RNA vectors. The recombinant vectors are preferably DNA plasmids or viral vectors. Ribozyme expressing viral vectors could be constructed based on, but not limited to, adeno-associated virus, retrovirus, adenovirus, or alphavirus. Preferably, the recombinant vectors capable of expressing the ribozymes are delivered as described below, and persist in target cells. Alternatively, viral vectors may be used that provide for transient expression of ribozymes. Such vectors can be repeatedly administered as necessary. Once expressed, the ribozymes cleave the target mRNA. Delivery of ribozyme expressing vectors can be systemic, such as by intravenous or intramuscular administration, by administration to target cells ex-planted from the patient followed by reintroduction into the patient, or by any other means that would allow for introduction into the desired target cell (for a review, see, Couture and Stinchcomb, 1996, TIG., 12, 510).
As used in herein xe2x80x9ccellxe2x80x9d is used in its usual biological sense, and does not refer to an entire multicellular organism, e.g., specifically does not refer to a human. The cell may be present in a non-human multicellular organism, e.g., birds, plants and mammals such as cows, sheep, apes, monkeys, swine, dogs, and cats.
In a preferred embodiment, an expression vector comprising a nucleic acid sequence encoding at least one of the nucleic acid catalysts of the instant invention is disclosed. The nucleic acid sequence encoding the nucleic acid catalyst of the instant invention is operable linked in a manner which allows expression of that nucleic acid molecule.
In one embodiment, the expression vector comprises: a transcription initiation region (e.g., eukaryotic pol I, II or III initiation region); b) a transcription termination region (e.g., eukaryotic pol I, II or III termination region); c) a nucleic acid sequence encoding at least one of the nucleic acid catalyst of the instant invention; and wherein said gene is operably linked to said initiation region and said termination region, in a manner which allows expression and/or delivery of said nucleic acid molecule. The vector may optionally include an open reading frame (ORF) for a protein operably linked on the 5xe2x80x2 side or the 3xe2x80x2-side of the sequence encoding the nucleic acid catalyst of the invention; and/or an intron (intervening sequences).
By xe2x80x9cpatientxe2x80x9d is meant an organism which is a donor or recipient of explanted cells or the cells themselves. xe2x80x9cPatientxe2x80x9d also refers to an organism to which enzymatic nucleic acid molecules can be administered. Preferably, a patient is a mammal or mammalian cells. More preferably, a patient is a human or human cells.
By xe2x80x9cvectorsxe2x80x9d is meant any nucleic acid- and/or viral-based technique used to deliver a desired nucleic acid.
Another means of accumulating high concentrations of a ribozyme(s) within cells is to incorporate the ribozyme-encoding sequences into a DNA or RNA expression vector. Transcription of the ribozyme sequences are driven from a promoter for eukaryotic RNA polymerase. I (pol I), RNA polymerase II (pol II), or RNA polymerase III (pol III). Transcripts from pol II or pol III promoters will be expressed at high levels in all cells; the levels of a given pol II promoter in a given cell type will depend on the nature of the gene regulatory sequences (enhancers, silencers, etc.) present nearby. Prokaryotic RNA polymerase promoters are also used, providing that the prokaryotic RNA polymerase enzyme is expressed in the appropriate cells (Elroy-Stein and Moss, 1990 Proc. Natl. Acad. Sci. USA, 87, 6743-7; Gao and Huang 1993 Nucleic Acids Res., 21, 2867-72; Lieber et al., 1993 Methods Enzymol., 217, 47-66; Zhou et al., 1990 Mol. Cell. Biol., 10, 4529-37). Several investigators have demonstrated that ribozymes expressed from such promoters can function in mammalian cells (e.g. Kashani-Sabet et al., 1992 Antisense Res. Dev., 2, 3-15; Ojwang et al., 1992 Proc. Natl. Acad. Sci. USA, 89, 10802-6; Chen et al., 1992 Nucleic Acids Res., 20, 4581-9; Yu et al., 1993 Proc. Natl. Acad. Sci. USA, 90, 6340-4; L""Huillier et al., 1992 EMBO J. 11, 4411-8; Lisziewicz et al., 1993 Proc. Natl. Acad. Sci. U. S. A., 90, 8000-4; Thompson et al., 1995 Nucleic Acids Res. 23, 2259; Sullenger and Cech, 1993, Science262, 1566). The above ribozyme transcription units can be incorporated into a variety of vectors for introduction into mammalian cells, including but not restricted to, plasmid DNA vectors, viral DNA vectors (such as adenovirus or adeno-associated virus vectors), or viral RNA vectors (such as retroviral or alphavirus vectors) (for a review see Couture and Stinchcomb, 1996, supra).
In another preferred embodiment, catalytic activity of the molecules described in the instant invention can be optimized as described by Draper et al., supra. The details will not be repeated here, but include altering the length of the ribozyme binding arms, or chemically synthesizing ribozymes with modifications (base, sugar and/or phosphate) that prevent their degradation by serum ribonucleases and/or enhance their enzymatic activity (see e.g., Eckstein et al., International Publication No. WO 92/07065; Perrault et al., 1990 Nature 344, 565; Pieken et al., 1991 Science 253, 314; Usman and Cedergren, 1992 Trends in Biochem. Sci. 17, 334; Usman et. al., International Publication No. WO 93/15187; and Rossi et al., International Publication No. WO 91/03162; Sproat, U.S. Pat. No. 5;334,711; and Burgin et al., supra; all of these describe various chemical modifications that can be made to the base, phosphate and/or sugar moieties of enzymatic RNA molecules). Modifications to the molecules, which enhance their efficacy in cells, and removal of bases from stem loop structures to shorten RNA synthesis times and reduce chemical requirements are desired. All these publications are hereby incorporated by reference herein.
By xe2x80x9cenhanced enzymatic activityxe2x80x9d is meant to include activity measured in cells and/or in vivo where the activity is a reflection of both catalytic activity and ribozyme stability. In this invention, the product of these properties is increased or not significantly (less than 10-fold) decreased in vivo compared to an all RNA ribozyme.
In yet another preferred embodiment, nucleic acid catalysts having chemical modifications, which maintain or enhance enzymatic activity are provided. Such nucleic acid is also generally more resistant to nucleases than unmodified nucleic acid. Thus, in a cell and/or in vivo the activity may not be significantly lowered. As exemplified herein, such ribozymes are useful in a cell and/or in vivo even if activity over all is reduced 10 fold (Burgin et al., 1996, Biochemistry, 35, 14090). Such ribozymes herein are said to xe2x80x9cmaintainxe2x80x9d the enzymatic activity of an all RNA ribozyme.
In yet another aspect the invention features an enzymatic nucleic acid molecule which cleaves mRNA produced from the human K-ras gene (accession NMxe2x80x94004985), H-ras gene (accession NMxe2x80x94005343) or N-ras gene (accession NMxe2x80x94002524). In one nonlimiting example, the enzymatic nucleic acid molecule of the present invention cleaves mRNA molecules associated with the development or maintenance of colon carcinoma. In preferred embodiments, the enzymatic nucleic acid molecules comprise sequences which are complementary to the substrate sequences in Table II. Examples of such enzymatic nucleic acid molecules also are shown in Table II. Examples of such enzymatic nucleic acid molecules consist essentially of sequences defined in Table II.
Other features and advantages of the invention will be apparent from the following description of the preferred embodiments thereof, and from the claims.
The drawings will first briefly be described.
FIG. 1 A) is a diagrammatic representation of the hammerhead ribozyme domain known in the art. Stem II can be 2 base-pairs long. Each N is independently any base or non-nucleotide as used herein; B) is a diagrammatic representation of a self-cleaving hammerhead ribozyme; C) is a diagrammatic representation of a random pool of self-cleaving RNA. N indicates the region with random nucleotides. (SEQ ID NO: 1171-1174)
FIG. 2 is a schematic representation of a non-limiting in vitro selection strategy used to evolve nucleic acid catalysts. (SEQ ID NO: 1175-1189)
FIG. 3 shows sequences of individual RNAs that represent new classes of self-cleaving ribozymes. Also shown are rates of catalysis (kin-cis (minxe2x88x921)) for each of the new self-cleaving RNA sequences. The underlined sequences indicate the regions capable of interacting with each other. (SEQ ID NO: 1175-1189)
FIG. 4 summarizes structure mapping studies on clone 40 RNA. (SEQ ID NO: 1190)
FIG. 5 shows sequence and possible secondary structure of a trans-cleaving ribozyme of the invention. Arrow identifies the site of cleavage. (SEQ ID NO: 1191-1192)
FIG. 6 shows non-limiting examples of trans-cleaving ribozyme-substrate complex of the present invention. (SEQ ID NO: 1193-1198)
FIG. 7 shows non-limiting examples of chemically modified trans-cleaving ribozyme-substrate complex of the present invention. (SEQ ID NO: 1199-1206)
FIG. 8 shows a non-limiting example of a chemically modified trans-cleaving ribozyme-substrate complex of the present invention and the numbering system used for (SEQ ID NO: 1207-1208)